Pharmaceutical compositions for combination therapy

ABSTRACT

This invention relates to the use of pharmaceutical compositions comprising a therapeutically effective combination of Tesofensine and Metoprolol for preventing the cardiovascular side effects of Tesofensine, while leaving the robust inhibitory efficacy on food intake and body weight loss unaffected.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/206,477, fled Jul. 11, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/936,963, filed Nov. 10, 2015, now U.S. Pat. No.9,387,184, which is a continuation of U.S. patent application Ser. No.14/379,032, filed Aug. 15, 2014, now U.S. Pat. No. 9,211,271, which isthe national stage entry of International Patent Application No.PCT/EP2013/052941, filed Feb. 14, 2013, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 61/599,623, filedFeb. 16, 2012, and Denmark Application No. PA201270076, filed Feb. 16,2012, the entireties of which are incorporated herein by reference.

TECHNICAL FIELD

This invention relates to the use of pharmaceutical compositionscomprising a therapeutically effective combination of Tesofensine andMetoprolol for preventing the cardiovascular side effects ofTesofensine, while leaving the robust inhibitory efficacy on food intakeand body weight loss unaffected.

BACKGROUND ART

Within the past decades the prevalence of obesity has risen in virtuallyall ethnic, racial and socioeconomic populations, in both genders and inall age groups. Obesity is associated with a significantly elevated riskfor type 2 diabetes, coronary heart diseases, hypertension and numerousother major illnesses and overall mortality from all causes. Therefore,weight reduction is critical for the obese patient. Thus there isimpetus for creating new and alternative treatments for management ofobesity.

Tesofensine, i.e.(1R,2R,3S,5S)-3-(3,4-dichlorophenyl)-2-(ethoxymethyl)-8-methyl-8-azabicyclo[3.2.1]octane],first described in WO 97/30997, is a triple monoamine reuptake inhibitorin development for the treatment of obesity.

WO 2005/070427 describes the use of certain monoamine neurotransmitterre-uptake inhibitors for obtaining a sustained reduction of body weight.WO 2009/065845 describes the use of certain monoamine neurotransmitterre-uptake inhibitors for the treatment of over-eating disorders. WO2009/080691 describes the use of certain monoamine neurotransmitterre-uptake inhibitors in a combination with additional anti-obesityagents for the treatment of obesity.

Tesofensine effectively produces a weight loss in obese individuals ofabout twice of that seen with currently marketed anti-obesity drugs.Results from clinical studies with Tesofensine also showed that thecompound has a good safety profile and is well tolerated. However,though no clinically relevant cardiovascular adverse events or changesin either blood pressure or pulse were seen, some cardiovascular effectswere measured with slight increases in heart rate and trends in bloodpressure. Although such small effects have no immediate risk to thepatient, some medical and regulatory concerns have been raised based onobservational studies, that even small changes in cardiovascularparameters may have long term implications on patients' benefit/riskevaluation.

Preclinical and clinical data suggest that appetite suppression is animportant mechanism by which Tesofensine exerts its robust weightreducing effect. Notably, the strong hypophagic response (i.e. lessappetite, decreased feeding) to Tesofensine treatment is demonstrated tobe linked to central stimulation of noradrenergic and dopaminergicneurotransmission. However, the sympathomimetic mode of action ofTesofensine may also associate with the elevated heart rate and bloodpressure observed in clinical settings.

WO 2009/080693 describes pharmaceutical compositions comprising certainmonoamine neurotransmitter re-uptake inhibitors in a combination withcertain beta blockers, and WO 2011/100659 describes a method forameliorating drug-induced elevation of blood pressure or increase inheartbeat by administration of an antihypertensive drug.

As such combination therapies seem tempting, drug combinations ofTesofensine with antihypertensive agents representing differentmechanisms of action have been investigated. Based on these experimentsit was found that some antihypertensive agents actually happen tointerfere with the anti-obesity effects of Tesofensine, and thus are notsuited for such combination therapy. Moreover, other antihypertensiveagents are actually unable to reverse the increase in systolic bloodpressure and heart rate induced by Tesofensine.

Metoprolol, i.e.1-(Isopropylamino)-3-[4-(2-methoxyethyl)-phenoxy]-propan-2-ol, brandedunder various trade names, is a selective β1 (adrenergic) receptorblocker normally used in the treatment of various disorders of thecardiovascular system, and in particular hypertension.

SUMMARY OF THE INVENTION

It has now surprisingly been found that the use of Metoprolol, in aspecific combination therapy with Tesofensine, for the treatment ofobesity, shows promising results in terms of preventing thecardiovascular effects induced Tesofensine, while leaving the robustinhibitory efficacy of Tesofensine on food intake and body weight lossunaffected.

Therefore, in one aspect, the invention provides a method of treatment,prevention or alleviation of obesity, or an obesity associated disorder,and for treatment, prevention or alleviation of the cardiovascular sideeffects of Tesofensine, in a living animal body, including a human,which method comprises the step of administering to such a living animalbody in need thereof, a therapeutically effective amount of Tesofensine,or a pharmaceutically acceptable salt thereof; in a combination therapywith Metoprolol, or a pharmaceutically acceptable salt thereof.

In another aspect the invention provides a combination of Tesofensine,or a pharmaceutically acceptable salt thereof, and Metoprolol, or apharmaceutically acceptable salt thereof, the treatment, prevention oralleviation of obesity, or an obesity associated disorder, and fortreatment, prevention or alleviation of the cardiovascular side effectsof Tesofensine.

In a third aspect the invention provides a combination of Tesofensine,or a pharmaceutically acceptable salt thereof, and Metoprolol, or apharmaceutically acceptable salt thereof, for use as a medicament.

In a fourth aspect the invention provides a combination of Tesofensine,or a pharmaceutically acceptable salt thereof; and Metoprolol, or apharmaceutically acceptable salt thereof; for the treatment, preventionor alleviation of obesity, or an obesity associated disorder, and fortreatment, prevention or alleviation of the cardiovascular side effectsof Tesofensine, in a mammal, including a human.

In a fifth aspect the invention relates to the use of a combination ofTesofensine, or a pharmaceutically acceptable salt thereof; andMetoprolol, or a pharmaceutically acceptable salt thereof; for themanufacture of a medicament for the treatment, prevention or alleviationof obesity, or an obesity associated disorder, and for treatment,prevention or alleviation of the cardiovascular side effects ofTesofensine, in a mammal, including a human.

In a sixth aspect the invention provides a pharmaceutical compositioncomprising Tesofensine, or a pharmaceutically acceptable salt thereof,for use in a combination therapy together with a pharmaceuticalcomposition comprising Metoprolol, or a pharmaceutically acceptable saltthereof, for the treatment, prevention or alleviation of obesity, or anobesity associated disorder, and for treatment, prevention oralleviation of the cardiovascular side effects of Tesofensine.

In an eight aspect the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of Tesofensine, or apharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Metoprolol, or a pharmaceutically acceptable saltthereof, together with one or more adjuvants, excipients, carriersand/or diluents.

In a ninth aspect the invention provides a kit of parts comprising atleast two separate unit dosage forms (A) and (B), wherein (A) comprisesTesofensine, or a pharmaceutically acceptable salt thereof; and (B)comprises Metoprolol, or a pharmaceutically acceptable salt thereof; andoptionally (C) instructions for the simultaneous, sequential or separateadministration of the Tesofensine of (A) and the Metoprolol of (B), to apatient in need thereof.

Other objects of the invention will be apparent to the person skilled inthe art from the following detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawing, in which:

FIGS. 1A-1E show that Tesofensine (given 1.0-5.0 mg/kg, p.o.)dose-dependently inhibits food intake, reduces body weight, andstimulates locomotor activity in telemetrized rats. FIG. 1A depicts meancumulative food intake (2 hour intervals) measured over 48 hours afteracute dosing of tesofensine or saline vehicle. FIG. 1B depicts mean foodintake measured over 48 hours, averaged in the 12 hours dark and lightphase intervals, respectively. FIGS. 1C and 1D depict net body weightgain given relative (%) and absolute values (grams), respectively,compared to body weight measured prior to dosing on day 0. FIG. 1Edepicts mean locomotor activity measured over 48 hours, averaged in thecorresponding 12 hours dark and light phase intervals, respectively.Dark and white horizontal bars below the x-axis indicate 12 hours darkand light phases, respectively. Abbreviations: VEH, vehicle; TESO,tesofensine. *p<0.05, **p<0.01, ***p<0.001 (compared to VEH)];

FIGS. 2A-2C-show that Tesofensine (given 1.0-5.0 mg/kg, p.o.)dose-dependently elevates heart rate and blood pressure in telemetrizedrats. FIG. 2A depicts mean heart rate ±S.E.M. averaged in 12 hoursintervals. FIG. 2B depicts mean diastolic blood pressure averaged in 12hours intervals. FIG. 2C depicts mean systolic blood pressure averagedin 12 hours intervals. Dark and white horizontal bars below the x-axisindicate 12 hours dark and light phases, respectively. Abbreviations:VEH, vehicle; TESO, tesofensine; bpm, beats per minute. *p<0.05,**p<0.01, ***p<0.001 (compared to VEH)];

FIGS. 3A-3E show dose-response effects of Metoprolol (given 10, 20mg/kg, p.o.) in combination with Tesofensine (3.0 mg/kg, p.o.), and thatMetoprolol does not affect the hypophagic and weight-lowering effects oftesofensine, but blocks tesofensine-induced locomotor activity intelemetrized rats. FIG. 3A depicts mean cumulative food intake (2 hoursintervals) measured over 48 hours after acute administration ofMetoprolol+Tesofensine combinations or saline vehicle. FIG. 3B depictsmean food intake measured over 48 hours, averaged in the 12 hours darkand light phase intervals, respectively. FIGS. 3C and 3D depict net bodyweight gain given in relative (%) and absolute values (grams) comparedto body weight measured prior to dosing on day 0. FIG. 3E depicts meanlocomotor activity measured over 48 hours, averaged in the 12 hours darkand light phase intervals, respectively. Dark and white horizontal barsbelow the x-axis indicate 12 hours dark and light phases, respectively.Abbreviations: VEH, vehicle, TESO, tesofensine; MET, Metoprolol.**p<0.01, ***p<0.001 (compared to VEH+VEH); #p<0.05, ##p<0.01 (comparedto VEH+TESO 3.0)];

FIGS. 4A-4C show that Metoprolol dose-dependently blockstesofensine-induced rise in heart rate and blood pressure intelemetrized rats. FIG. 4A depicts mean heart rate ±S.E.M. averaged in12 hours intervals. FIG. 4B depicts mean diastolic blood pressureaveraged in 12 hours intervals. FIG. 4C depicts mean systolic bloodpressure averaged in 12 hours intervals. Dark and white horizontal barsbelow the x-axis indicate 12 hours dark and light phases, respectively.Abbreviations: VEH, vehicle; TESO, tesofensine; MET, Metoprolol.**p<0.01, ***p<0.001 (compared to VEH+VEH); #p<0.05, ##p<0.01,###p<0.001 (compared to VEH+TESO 3.0)];

FIGS. 5A-5E show dose-response effects of Telmisartan (1.0, 3.0 mg/kg,p.o.) in combination with tesofensine (3.0 mg/kg, p.o.), and thatTelmisartan does not affect the hypophagic and weight-lowering effectsof Tesofensine, and has no effect on tesofensine-induced locomotoractivity in telemetrized rats. FIG. 5A depicts mean cumulative foodintake (2 hours intervals) measured over 48 hours after acuteadministration of Telmisartan+Tesofensine combinations or salinevehicle. FIG. 5B depicts mean food intake measured over 48 hours,averaged in the 12 hours dark and light phase intervals, respectively.FIGS. 5C and 5D depict net body weight gain given in relative (%) andabsolute values (grams) compared to body weight measured prior to dosingon day 0. FIG. 5E depicts mean locomotor activity measured over 48hours, averaged in the 12 hours dark and light phase intervals,respectively. Dark and white horizontal bars below the x-axis indicate12 hours dark and light phases, respectively. Abbreviations: VEH,vehicle; TESO, tesofensine; TEL, Telmisartan. *p<0.05, **p<0.01,***p<0.001 (compared to VEH+VEH)]; and

FIGS. 6A-6C show effects of combined Tesofensine+Telmisartan drugtreatment on heart rate and blood pressure in telemetrized rats. FIG. 6Adepicts mean heart rate ±S.E.M. averaged in 12 hours intervals. FIG. 6Bdepicts mean diastolic blood pressure averaged in 12 hours intervals.FIG. 6C depicts mean systolic blood pressure averaged in 12 hoursintervals. Dark and white horizontal bars below the x-axis indicate 12hours dark and light phases, respectively. Dark and white horizontalbars below the x-axis indicate 12 hours dark and light phases,respectively. Abbreviations: VEH, vehicle; TESO, tesofensine; TEL,Telmisartan. *p<0.05, **p<0.01, ***p<0.001 (compared to VEH+VEH);#p<0.05 (compared to VEH+TESO 3.0)].

DETAILED DISCLOSURE OF THE INVENTION

Tesofensine is a centrally acting triple monoamine reuptake inhibitor(MRI) with intrinsic inhibitory activity on noradrenaline, serotonin anddopamine transporter function. When corrected for placebo and dieteffects, long-term Tesofensine treatment produces a weight loss of about10% in obese patients, which is twice as much as that achieved bycurrently marketed anti-obesity drugs.

The anti-obesity effect of Tesofensine is likely explained bydose-dependent hypophagia due to stimulation of satiety, suggesting thatTesofensine acts as an appetite suppressant to produce a negative energybalance. In addition, Tesofensine is also demonstrated to increasenocturnal energy expenditure in human subjects. These findings haverecently been corroborated and extended in preclinical settings,demonstrating that Tesofensine induces a robust and sustained weightloss in a rat model of diet-induced obesity (DIO) of which thelong-lasting drop in body weight is caused by appetite suppression witha gradually increase in energy expenditure. Notably, the hypophagiceffect of Tesofensine in DIO rats is critically dependent on stimulatedal adrenoceptor activity, and to a less extend dopamine D1 receptorfunction, indicating that enhancement of central noradrenergic anddopaminergic neurotransmission constitute important mechanismsunderlying the robust appetite-suppressing effect of Tesofensine.

Overall, chronic Tesofensine treatment is associated with minor adverseevents, and with minimal cardiovascular effects, suggesting thatTesofensine may be a well-tolerated long-term treatment for obesity.However, in this regard, dose-dependent elevations in heart rate andsignificant increases in blood pressure at the highest dose tested havebeen reported in obese individuals. We therefore speculated whether thesympathomimetic effects of Tesofensine might also associate with thereported effects on cardiovascular function.

To address this hypothesis, we simultaneously monitored effects on foodintake and body weight regulation in conjunction with cardiovascularparameters in telemetrized (i.e. spontaneously hypertensive) ratsfollowing administration of Tesofensine alone, or in drug combinationsof Tesofensine with antihypertensive agents representing differentmechanisms of action, i.e. Metoprolol (i.e. a β1 adrenoceptorantagonist) and Telmisartan (i.e. an angiotension AT1 receptorantagonist).

It has now surprisingly been found that use of Metoprolol, in a specificcombination therapy with Tesofensine, for the treatment of obesity orobesity associated disorders, shows promising results in terms ofpreventing the cardiovascular effects induced Tesofensine, while leavingthe robust inhibitory efficacy of Tesofensine on food intake and bodyweight loss unaffected.

Therefore, in one aspect, the present invention relates to a combinationtherapy using Tesofensine and Metoprolol for the treatment, preventionor alleviation of obesity or an obesity associated disorder in a subjectsuffering from such disorders.

In another aspect the invention relates to the of a combination ofTesofensine, or a pharmaceutically acceptable salt thereof; andMetoprolol, or a pharmaceutically acceptable salt thereof; for themanufacture of a medicament for the treatment, prevention or alleviationof obesity, or an obesity associated disorder, and for treatment,prevention or alleviation of the cardiovascular side effects ofTesofensine, in a mammal, including a human.

Obesity and Obesity Associated Disorders

Obesity is a medical condition in which excess body fat has accumulatedto the extent that it may have an adverse effect on health, leading toreduced life expectancy and/or increased health problems. Body massindex (BMI), a measurement which compares weight and height, definespeople as overweight (pre-obese) if their BMI is between 25 and 30kg/m², and obese when it is greater than 30 kg/m².

In a preferred embodiment, the combination therapy according to theinvention is contemplated useful for the treatment of pre-obesesubjects, i.e. having a BMI between 25 and 30 kg/m².

In another preferred embodiment, the combination therapy according tothe invention is contemplated useful for the treatment of obesesubjects, i.e. having a BMI of above 30 kg/m².

In a third preferred embodiment, the combination therapy according tothe invention is contemplated useful for the treatment of morbid obesesubjects, i.e. having a BMI of above 35 kg/m².

In the context of this invention an obesity associated disorder is adisorder or condition selected from the group consisting of over-eatingdisorders, bulimia nervosa, binge eating disorder, compulsiveover-eating, impaired appetite regulation, metabolic syndrome, type 2diabetes, dyslipidemia, atherosclerosis and drug-induced obesity, e.g.following therapy with antidepressive or antipsychotic drugs.

Pharmaceutically Acceptable Salts

The active compounds for use according to the invention may be providedin any form suitable for the intended administration. Suitable formsinclude pharmaceutically (i.e. physiologically) acceptable salts, andpre- or prodrug forms of the compound of the invention.

Examples of pharmaceutically acceptable addition salts include, withoutlimitation, the non-toxic inorganic and organic acid addition salts suchas the hydrochloride, the hydrobromide, the nitrate, the perchlorate,the phosphate, the sulphate, the formate, the acetate, the aconate, theascorbate, the benzenesulphonate, the benzoate, the cinnamate, thecitrate, the embonate, the enantate, the fumarate, the glutamate, theglycolate, the lactate, the maleate, the malonate, the mandelate, themethanesulphonate, the naphthalene-2-sulphonate, the phthalate, thesalicylate, the sorbate, the stearate, the succinate, the tartrate, thetoluene-p-sulphonate, and the like. Such salts may be formed byprocedures well known and described in the art.

Examples of pharmaceutically acceptable cationic salts of a chemicalcompound of the invention include, without limitation, the sodium, thepotassium, the calcium, the magnesium, the zinc, the aluminium, thelithium, the choline, the lysinium, and the ammonium salt, and the like,of a chemical compound of the invention containing an anionic group.Such cationic salts may be formed by procedures well known and describedin the art.

In the context of this invention the “onium salts” of N-containingcompounds are also contemplated as pharmaceutically acceptable salts.Preferred “onium salts” include the alkyl-onium salts, thecycloalkyl-onium salts, and the cycloalkylalkyl-onium salts.

Examples of pre- or prodrug forms of the chemical compound for useaccording to the invention include examples of suitable prodrugs of thesubstances for use according to the invention include compounds modifiedat one or more reactive or derivatizable groups of the parent compound.Of particular interest are compounds modified at a carboxyl group, ahydroxyl group, or an amino group. Examples of suitable derivatives areesters or amides.

The chemical compounds for use according to the invention may beprovided in dissoluble or indissoluble forms together with apharmaceutically acceptable solvent such as water, ethanol, and thelike. Dissoluble forms may also include hydrated forms such as themonohydrate, the dihydrate, the hemihydrate, the trihydrate, thetetrahydrate, and the like. In general, the dissoluble forms areconsidered equivalent to indissoluble forms for the purposes of thisinvention.

Pharmaceutical Compositions

In another aspect the invention relates to a combination of Tesofensine,or a pharmaceutically acceptable salt thereof, and Metoprolol, or apharmaceutically acceptable salt thereof, for use as a medicament.

In a further aspect the invention provides pharmaceutical compositionscomprising Tesofensine, or a pharmaceutically acceptable salt thereof,for use in a combination therapy together with a pharmaceuticalcomposition comprising Metoprolol, or a pharmaceutically acceptable saltthereof, for the treatment, prevention or alleviation of obesity, or anobesity associated disorder, and for treatment, prevention oralleviation of the cardiovascular side effects of Tesofensine.

In a yet further aspect the invention provides pharmaceuticalcompositions comprising a therapeutically effective amount ofTesofensine, or a pharmaceutically acceptable salt thereof, and atherapeutically effective amount of Metoprolol, or a pharmaceuticallyacceptable salt thereof, together with one or more adjuvants,excipients, carriers and/or diluents.

While the compounds for use according to the invention may beadministered in the form of the raw compound, it is preferred tointroduce the active ingredients, optionally in the form ofphysiologically acceptable salts, in a pharmaceutical compositiontogether with one or more adjuvants, excipients, carriers, buffers,diluents, and/or other customary pharmaceutical auxiliaries.

In a preferred embodiment, the invention provides pharmaceuticalcompositions comprising the active compounds or pharmaceuticallyacceptable salts or derivatives thereof, together with one or morepharmaceutically acceptable carriers therefore, and, optionally, othertherapeutic and/or prophylactic ingredients, know and used in the art.The carrier(s) must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not harmful to therecipient thereof.

The pharmaceutical composition of the invention may be administered byany convenient route, which suits the desired therapy. Preferred routesof administration include oral administration, in particular in tablet,in capsule, in dragé, in powder, or in liquid form, and parenteraladministration, in particular cutaneous, subcutaneous, intramuscular, orintravenous injection. The pharmaceutical composition of the inventioncan be manufactured by the skilled person by use of standard methods andconventional techniques appropriate to the desired formulation. Whendesired, compositions adapted to give sustained release of the activeingredient may be employed.

Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

The dosage of the compound of Formula I is determined as the API (ActivePharmaceutical Ingredient), i.e. calculated as the free base. The actualdosage of each of the active ingredients depends on the nature andseverity of the disease being treated, the exact mode of administration,form of administration and is within the discretion of the physician,and may be varied by titration of the dosage to the particularcircumstances of this invention to produce the desired therapeuticeffect.

It is currently believed that a daily dosage of Tesofensine in the rangeof from about 0.1 to about 1 mg active ingredient, preferably of fromabout 0.1 to about 0.5 mg active ingredient, is suitable for therapeutictreatments. The daily dosage of Tesofensine may be administered in oneor several doses, such as two, per day. In one embodiment, the dailydosage is administered in one dose.

The daily dosage of Metoprolol is presently contemplated to be in therange of from about 25 to about 200 mg of active ingredient, preferablyof from about 25 to about 100 mg active ingredient. The daily dosage ofMetoprolol may be administered in one or several doses, such as two, perday. In one embodiment, the daily dosage is administered in one dose.

Pharmaceutical Kits of Parts

According to the invention there is also provided a kit of partscomprising at least two separate unit dosage forms (A) and (B), wherein

(A) comprises Tesofensine, or a pharmaceutically acceptable saltthereof; and

(B) comprises Metoprolol, or a pharmaceutically acceptable salt thereof;and optionally

(C) instructions for the simultaneous, sequential or separateadministration of the Tesofensine of (A) and the Metoprolol of (B), to apatient in need thereof.

Tesofensine for use according to the invention and Metoprolol for useaccording to the invention may preferably be provided in a form that issuitable for administration in conjunction with the other. This isintended to include instances where one or the other of two formulationsmay be administered (optionally repeatedly) prior to, after, and/or atthe same time as administration with the other component.

Also Tesofensine for use according to the invention and Metoprolol foruse according to the invention may be administered in a combined form,or separately or separately and sequentially, wherein the sequentialadministration is close in time or remote in time. This may inparticular include that two formulations are administered (optionallyrepeatedly) sufficiently closely in time for there to be a beneficialeffect for the patient, that is greater over the course of the treatmentof the relevant condition than if either of the two formulations areadministered (optionally repeatedly) alone, in the absence of the otherformulation, over the same course of treatment. Determination of whethera combination provides a greater beneficial effect in respect of, andover the course of treatment of, a particular condition, will dependupon the condition to be treated or prevented, but may be achievedroutinely by the person skilled in the art.

When used in this context, the terms “administered simultaneously” and“administered at the same time as” include that individual doses ofTesofensine and are administered within 48 hours, e.g. 24 hours, of eachother.

Bringing the two components into association with each other, includesthat components (A) and (B) may be provided as separate formulations(i.e. independently of one another), which are subsequently broughttogether for use in conjunction with each other in combination therapy;or packaged and presented together as separate components of a“combination pack” for use in conjunction with each other in combinationtherapy.

Methods of Therapy

In another aspect the invention provides methods of treatment,prevention or alleviation of obesity or an obesity associated disease ofa living animal body, including a human, which method comprises the stepof administering to such a living animal body in need thereof, atherapeutically effective amount of a combination of Tesofensine, or apharmaceutically acceptable salt thereof, and Metoprolol, apharmaceutically acceptable salt thereof.

In a preferred embodiment the obesity associated disorder is a disorderor condition selected from the group consisting of over-eatingdisorders, bulimia nervosa, binge eating disorder, compulsiveover-eating, impaired appetite regulation, metabolic syndrome, type 2diabetes, dyslipidemia, atherosclerosis and drug-induced obesity.

It is currently believed that a daily dosage of Tesofensine in the rangeof from about 0.1 to about 1 mg active ingredient, preferably of fromabout 0.1 to about 0.5 mg active ingredient, is suitable for therapeutictreatments. The daily dosage of Tesofensine may be administered in oneor several doses, such as two, per day. In one embodiment, the dailydosage is administered in one dose.

The daily dosage of Metoprolol is presently contemplated to be in therange of from about 25 to about 200 mg of active ingredient, preferablyof from about 25 to about 100 mg active ingredient. The daily dosage ofMetoprolol may be administered in one or several doses, such as two, perday. In one embodiment, the daily dosage is administered in one dose.

EXAMPLES

The invention is further illustrated with reference to the followingexample, which is not intended to be in any way limiting to the scope ofthe invention as claimed.

Animal Care and Housing

Five months-old male normotensive Sprague-Dawley rats (508±18 g, Harlan,Horst, The Netherlands) were housed in solid bottomed Plexiglas cageswith dust free wood chippings and a cardboard tube. Holding rooms weremaintained under a 12-h light/dark cycle (lights off: 1500 h). Ambienttemperature was 18.0 to 22.0° C. and relative air humidity of 40 to 60%.A dim red light was the sole source of illumination during the darkperiod. The rats had ad libitum standard chow (Altromin 1324, 10% kcalfrom fat, energy density 2.85 kcal per g, Altromin GmbH, Lage, Germany)and water. All experiments were approved (permission no. 2007/561-1343)and conducted in accordance with the guidelines of the AnimalExperimentation Inspectorate, Ministry of Justice, Denmark.

Simultaneous Real-Time Feeding and Cardiovascular Monitoring inTelemetrized Rats Telemetry Set-Up

The rats were implanted at Harlan laboratories, Horst, the Netherlands,with Data Science International (DSI, St. Paul, USA) Physiotel PA-C40transmitters according to the manufactures description. In brief, therats were anaesthetized with isoflurane, ventilated, and a laparotomywas performed under aseptic conditions. A pressure catheter was insertedand sealed in place with Vetbond (3M, St. Paul, USA) into the isolatedabdominal aorta. Finally, the transmitter was placed on top of theintestines, in parallel to the long axis of the body, and secured to theabdominal wall, where after the abdominal muscle layer and skin wasclosed with solvable sutures. The animals were allowed fullpost-surgical recovery before shipment. Blood pressure (systolic anddiastolic arterial blood pressure) and heart rate (pulse rate) data werecollected at a sampling rate of 500 Hz using Dataquest A.R.T (v.4.3) andPonemah software (v.5.0) (DSI, St. Paul, USA) using factory-providedcalibration values for the individual transmitters and an AmbientPressure Reference Monitor (DSI, St. Paul, USA) to ensure accurate bloodpressure measurements. Data were collected continuously for 48 h andbinned in 5 s intervals.

Real-Time Feeding Monitoring in Telemetrized Rats

Upon 2-3 weeks of post-surgery recovery, the rats were transferred tofully automated food intake monitoring cages (HM-2, MBRose, Faaborg,Denmark) modified to simultaneously determine individualized food intake(by microchip, see below) and cardiovascular condition (by telemetry).For combined telemetry analysis, two receivers (RPC-1, Data SciencesInternational, St. Paul, Minn.) were placed in the bottom of each HM-2food intake monitoring cages thereby fully covering the cage surfacearea. The modified HM-2 food intake monitoring cages were placed in amodified ventilated cabinet with lightproof doors and a light kit forcabinet-based control of light-dark cycle (Scanbur BK, Karslunde,Denmark) being similar to that in the holding rooms. Cabinet temperaturewas 24.0 to 26.0° C. and relative air humidity of 40 to 60%. The animalswere habituated to the HM-2 food intake monitoring system for at least 5days prior to initiation of drug treatment procedures. Prior tore-housing to the fully automated food intake monitoring cages, the ratswere subcutaneously injected with a microchip (#402575, eVet, Haderslev,Denmark) to simultaneously identify, and in real-time mode to trackfeeding behavior of each individual animal throughout the entireexperiment. Locomotor activity was detected by an integrated infraredsensor placed above the cage. Standard HM-2 control unit settings arereported previously in further details⁷. All drugs and saline vehiclewere administered 30 min before dark onset. All rats received the sametreatment in each individual experiment, i.e. a parallel study designwas used, and a wash-out period of at least 5 days was used betweentreatments to assure reestablishment of baseline levels of food intake,locomotor activity, heart rate and blood pressure. The home cage wasremoved from the HM-2 food intake monitoring system during the drugadministration procedure and returned immediately after completion ofthe drug administration, whereupon automated monitoring of feedingbehavior and cardiovascular parameters of each individual animal wasresumed. Body weight was measured daily. Body weight and microstructuralfood intake analysis was performed using a data reporting software(HMView, MBRose, Faaborg, Denmark).

Statistics

Data were fed into a standard graphic and statistical analysis program(Graph Pad Prism v.4.03). Body weight data were calculated as absolutevalues (g) or daily body weight gain relative (control level=100%) tothe first day of drug administration (day 0). Body weight gain and foodintake were expressed as means±S.E.M of n individual animals. Afteracquisition of telemetry data, 12-hour means were calculated usingMicrosoft Excel 2007. Finally, statistical analysis and datapresentation (mean±S.E.M.) was performed using GraphPad Prism v.4.03).All data were evaluated using a repeated-measure one-way ANOVA withTukey's post-hoc test was applied to perform statistical comparisonsbetween treatment groups. A p-value less than 0.05 was consideredstatistically significant.

Drugs

Tesofensine(8-Azabicyclo[3.2.1]octane,3-(3,4-dichlorophenyl)-2-(ethoxymethyl)-8-methyl-[1R-(2-endo,3-exo)]-2-hydroxy-1,2,3-propanetricarboxylate)is a derivative of an azabicyclooctane citrate, synthesized at theDepartment of Medicinal Chemistry, NeuroSearch A/S.

Metoprolol and Telmisartan were purchased from Sigma (St. Louis, Mo.).Tesofensine and Metoprolol were dissolved in 0.9% saline solution,whereas Telmisartan was dissolved in 1 N NaOH and subsequently titratedwith 1 N HCl to pH 7.4.

All drugs were administered p.o. (1.0 ml/kg). In drug combinationexperiments, tesofensine and the anti-hypertensive drug wereadministered simultaneously (<1 min apart) as separate drug solutions.

Results Effects on Food Intake and Body Weight

Acute tesofensine administration robustly triggered a reduction of foodintake in telemetrized rats (FIGS. 1A and 1B). The food intake intesofensine-treated rats declined in a dose- and time-dependent fashionwith the highest oral dose (5.0 mg/kg) at 12 hours post-dosing reducingfood intake to approximately 50% of the control level (p<0.001). Thehypophagic effect of tesofensine was sustained for up to 12 hours (alldoses), 24 hours (3.0-5.0 mg/kg) and 48 hours (5.0 mg/kg) after dosing,respectively, whereupon food intake returned to baseline levels (FIG.1B). The hypophagic effect of tesofensine was paralleled by acorresponding dose-dependent reduction in body weight (negative bodyweight gain) with the highest doses (3.0-5.0 mg/kg) producing asignificant net body weight loss of 1.0-1.5% (equivalent to 8-11 g,compared to the body weight of vehicle-treated rats) being evident forat least 48 hours after drug administration (FIGS. 10 and 1D).Tesofensine also dose-dependently induced a significant, albeitshort-lasting, increase in locomotor activity in the dose-range of3.0-5.0 mg/kg (FIG. 1E).

The intermediate dose (3.0 mg/kg) of tesofensine was selected forfurther characterization in acute drug combination studies with theantihypertensive agents, Metoprolol (FIGS. 3A-3E) and Telmisartan (FIGS.5A-5E), respectively. These drug interaction studies indicated thatneither antihypertensive drug exhibited an effect on food intake or bodyweight regulation per se, and did also not affect tesofensine-inducedreductions in food intake (FIGS. 3A, 3B, 5A, 5B) and body weight (FIGS.3C, 3D, 5C, 5D). In contrast, Metoprolol (FIG. 3E, p<0.05 compared totesofensine alone), but not Telmisartan (FIG. 5E, p=0.98 compared totesofensine alone), completely prevented the locomotor activity inducingeffect by tesofensine.

Effects on Cardiovascular Parameters

As expected, the telemetric monitoring of blood pressure and heart rateshowed a clear diurnal variation (FIGS. 2A-2C), with higher bloodpressure and heart rate observed during the active (nocturnal) period.

Acute treatment with tesofensine caused a dose-dependent increase inheart rate at all doses tested, lasting for up to 48 hours aftertreatment (FIG. 2A). Similarly, a dose-dependent modest increase insystolic blood pressure was observed up to 48 hours after drugadministration (3.0 mg/kg and 5.0 mg/kg) (FIG. 2C). The effect of 3.0mg/kg tesofensine on heart rate and systolic blood pressure therebyoutlasted the hypophagic effects of 3.0 mg/kg tesofensine. A trendtowards a dose-dependent rise in diastolic blood pressure was alsoobserved, although the highest dose did not attain statisticalsignificance (p=0.204, FIG. 2B).

Two combination drug studies were carried out in order to investigate ifantihypertensive treatment could prevent or reduce the secondaryhypertension and elevated heart rate caused by tesofensine. Co-treatmentwith tesofensine (3.0 mg/kg) and the β₁ adrenoceptor antagonistMetoprolol (10 mg/kg and 20 mg/kg) fully reversed tesofensine-inducedtachycardia (FIG. 4A). The heart rate lowering effect of Metoprolol was,however, only observed during the first 24 hours after administration,whereas the heart rate was normalized to control levels in theMetoprolol+Tesofensine combination groups (FIG. 4A). The short-lastingeffects of Metoprolol reflect the pharmacokinetic properties in the rat.A normalization of the systolic blood pressure was also observed afterco-treatment with Metoprolol (20 mg/kg) for up to 24 hours (FIG. 4C).Similarly, the tesofensine-evoked increase in the diastolic pressureduring the first light phase (12-24 hours post-treatment) was reversedby co-treatment with Metoprolol (20 mg/kg, FIG. 4B). When administeredalone, Metoprolol (20 mg/kg) did not produce any significant effects ondiastolic blood pressure in the first 24 hours (FIGS. 4A-4C).

In a subsequent drug combinatorial study, tesofensine and theAT1-receptor antagonist Telmisartan was investigated. As for theMetoprolol study, a similar (3.0 mg/kg) dose of tesofensine was found tosignificantly increase heart rate. Co-treatment with Telmisartan (1.0and 3.0) did not revert the rise in heart rate after tesofensineadministration, and with the highest dose of Telmisartan combined withtesofensine we observed a significant increase in heart rate as comparedto tesofensine administration alone (FIG. 6A). Although co-treatmentwith Telmisartan was found to attenuate the increases in diastolic andsystolic blood pressure produced by Tesofensine, it did not lead to asignificant prevention of Tesofensine-induced hypertension (p>0.05,compared to Tesofensine alone, FIGS. 6B, 6C). Telmisartan alone (3.0mg/kg) had no effect on heart rate and blood pressure (FIGS. 6A-6C).

Discussion

Weight loss is often accompanied by an increase in perceived hunger andappetite sensations, which has been identified as an important predictorof weight relapse, and suppression of appetite function is thereforeconsidered very important for the maintenance of weight loss.

Recent clinical and preclinical reports have indicated that Tesofensineacts as a strong appetite suppressant by triggering satiety and fullnesssensations, which is believed to be a key mechanism underlying therobust anti-obesity effect of Tesofensine. Hence, the present data onTesofensine-induced anorexia in telemetrized rats further supports thisview. Tesofensine dose-dependently triggered a rapid hypophagic responselasting for up to 12-48 hours, depending on the dose administered. Thelong-lasting anorexigenic effect of Tesofensine suggests that thebioactive primary M1 metabolite (also being a triple MRI) of Tesofensinecontributed to the hypophagic and weight-lowering effect in rats, as theM1 metabolite exhibits significantly higher steady-state concentrationsand longer T_(1/2) in rodents.

In contrast, the human steady-state plasma concentrations of M1 areapproximately 60% lower as compared to those of Tesofensine, implyingthat the contribution of M1 to the overall activity might be lower inhumans. In addition, it is suggested that increased energy metabolismmay potentially contribute to the robust weight loss induced byTesofensine. Accordingly, a recent respiratory calorimetry studyindicated a moderate rise in fat oxidation and nocturnal thermogenesisafter short-term Tesofensine treatment in overweight or moderately obesemen. Also, while DIO rats show long-term sustained reductions in bodyweight during chronic Tesofensine treatment regimens, hypophagia is mostpronounced during the first week of treatment followed by a gradualdevelopment of tolerance to the anorexigenic effect of Tesofensine, thusbeing in indirect agreement with the clinical findings.

In the present study, Tesofensine dose-dependently increased locomotoractivity during the first 12 hours dark phase, and it may thus bepostulated that augmented locomotor activity may have contributed to theweight loss in telemetrized rats, e.g. by causing changes infood-seeking behavior or energy expenditure. However, the hypophagiceffect of Tesofensine was more potent and longer lasting (up to 48hours) as compared to the capacity of Tesofensine to induce locomotoractivity (up to 12 hours). In this regard, it is likely that thedifferent temporal pharmacodynamics on food intake and locomotoractivity is associated with the pharmacokinetics of Tesofensine.

In comparison to Tesofensine, the M1 metabolite has a longer T_(1/2)(see above) with a four- to five fold lower in vivo potency on dopaminereuptake transporter inhibition, which argues for the metabolite did notcontribute significantly to Tesofensine-induced locomotor activity.Also, the evidence that Metoprolol completely prevented the locomotorstimulatory effect of Tesofensine without affecting Tesofensine'sefficacy on hypophagia and body weight-reduction, indicates that themoderate increase locomotor activity did not promote a rise in energymetabolism. From these data we infer that locomotor effects had noinfluence on the appetite suppressing and weight loss effects ofTesofensine. In addition, it may be speculated that Metoprololantagonized Tesofensine-induced locomotor activity by indirect action onstriatal dopaminergic neurotransmission, as various β₁ blockers arereported to inhibit rat striatal dopamine release.

The preclinical finding of cardiovascular effects of Tesofensine inawake and freely moving rats is in accordance with clinical findings,also showing significant dose-dependent elevations in heart rate atlower dose levels than required to raise diastolic and systolic bloodpressure. Notably, the cardiovascular effects outlasted the hypophagiceffects following acute administration of Tesofensine. BecauseTesofensine and the M1 metabolite show equipotent inhibition ofnoradrenaline reuptake in vitro, it is likely that the M1 metabolitecontributed to the cardiovascular effects of Tesofensine.

Because β₁ adrenoceptor blockade by Metoprolol co-administration fullyprevented the cardiovascular effects of Tesofensine, this stronglyindicates that noradrenergic reuptake inhibitory component ofTesofensine is far the most important denominator for the cardiovascularadverse effects of Tesofensine.

Whether Tesofensine would affect blood pressure and heart ratedifferently in obese rats is not addressed in the present report andmust await further studies. It should also be noted that the presentobservations are restricted to the acute effects of Tesofensine, and donot exclude that the change in cardiovascular parameters in telemetrizedrats after chronic Tesofensine treatment may closer mimic clinicalfindings. Also being in good agreement with clinical and preclinicalreports, Tesofensine produced a strong hypophagic response with acorresponding body weight loss in telemetrized rats. Using normal-weightrats fed with chow, the efficacy and temporal pattern ofTesofensine-induced hypophagia and weight reduction observed in thepresent study is in accordance with similar findings in DIO rats,indicating that acute anti-obesity effects of Tesofensine can also bestudied in telemetrized non-obese rats.

Overall, the experimental in vivo settings used in the present study,allowing advanced synchronous monitoring of cardiovascular and foodintake parameters in real-time mode, represent a rational and validmethodology for simultaneously studying clinically relevant anti-obesityand vital sign effects of anti-obesity drugs.

Interestingly, the present results suggest a different pharmacodynamicprofile of Tesofensine+β₁ blocker combinational therapy as compared withSibutramine, a dual serotonin and noradrenaline reuptake inhibitor.Sibutramine exhibits a rather modest weight loss and significantelevates heart rate and blood pressure in obese patients, whichconstitutes a major concern in the clinical utility of Sibutramine.

A clinical study in obese hypertensive patients indicated thatSibutramine treatment with combined Ca²⁺ channel antagonist+ACEinhibitors or Metoprolol+hydrochlorothiazide treatment, respectively,significantly attenuated Sibutramine's anti-obesity effects. The lattercombination most negatively affected Sibutramine's weight-reducingefficacy which may be explained by the common observation thatβ-blockers can induce weight gain per se.

In contrast, Metoprolol therapy did not significantly interfere withSibutramine's anti-obesity and metabolic effects in a study onnormotensive obese patients, leaving it so far unresolved whethercombined β₁ blocker treatment is feasible to reduce cardiovascularadverse effects of Sibutramine in obese subjects. In this context, itshould be noted that anorexigenic effects of Sibutramine are believed tobe closely associated with stimulated α₁- and β₁-adrenoceptor function,as Sibutramine-induced hypophagia is antagonized by Prazosin andMetoprolol, respectively.

The implications from these studies may be that anti-obesity drugs withnoradrenergic activity will potentially have less anti-obesity efficacywhen combined with β-blockers to ameliorate any sympatheticcardiovascular effects. However, the present study suggests that thismay not be the case for Tesofensine, because combined treatment withMetoprolol did not affect the anti-obesity effects of Tesofensine.Hence, this observation indicate a clear pharmacodynamic separationbetween two distinct and important mechanisms of action of Tesofensine,namely the anti-obesity effects associated with β₁ adrenoceptorstimulation and cardiovascular effects linked to augmented β₁adrenoceptor function. The β₁ adrenoceptor effect of Tesofensine issuggested to be secondary to a blockade of hypothalamic synapticnoradrenaline reuptake leading to inhibition of intrahypothalamicappetite signaling circuits to evoke satiety responses.

In contrast, it is most conceivable that the cardiovascular effects ofTesofensine are being mediated via increased peripheral noradrenergictonus. Also being in contrast to Sibutramine, the anorexigenic effect ofTesofensine requires stimulation of both β₁ adrenoceptor and dopamine D₁receptor function to obtain full appetite-suppressing activity in DIOrats, hence indirectly pointing to the possibility that Tesofensinetreatment leads to recruitment of dopaminergic neurotransmission. Thisis relevant, as obese human subjects have indices of impaired centraldopaminergic activity thought to instigate overeating behavior tocompensate for a lowered hedonic drive.

In healthy human volunteers Tesofensine blocks the neuronal dopamineuptake transporter (DAT) at doses causing weight loss in obeseindividuals. This finding indicates that the dopamine enhancing effectof tesofensine is involved in mediating the weight reducing effect.

In conclusion, we demonstrate that combined Tesofensine and Metoprololtreatment preserves Tesofensine's anti-obesity efficacy while alsopreventing elevations in heart rate and blood pressure in rats. Thesefindings invite the possibility that combined antihypertensive treatmentwith Tesofensine would also be effective in obese patients.

1. A method of inducing hypophagia in a human comprising: administeringTesofensine, or a pharmaceutically acceptable salt thereof, to thehuman; in combination with Metoprolol, or a pharmaceutically acceptablesalt thereof, wherein the combination is effective in inducinghypophagia and wherein the combination is effective in preventing oralleviating drug-induced cardiovascular side-effects.
 2. The method ofclaim 1, wherein the Tesofensine, or pharmaceutically acceptable saltthereof, is administered at dosage of 0.1 mg to 1 mg daily, based on theamount of free base.
 3. The method of claim 1, wherein the Tesofensine,or pharmaceutically acceptable salt thereof, is administered at dosageof 0.1 mg to about 0.5 mg daily, based on the about of free base.
 4. Themethod of claim 1, wherein the Metoprolol, or pharmaceuticallyacceptable salt thereof, is administered at dosage of 0.25 mg to 200 mgdaily, based on the amount of free base.
 5. The method of claim 1,wherein the Metoprolol, or pharmaceutically acceptable salt thereof, isadministered at dosage of 25 mg to 100 mg daily, based on the amount offree base.
 6. The method of claim 1, wherein the human is a pre-obesehuman, an obese human, or a morbidly obese human.
 7. The method of claim1, wherein the human suffers from over-eating disorders, bulimianervosa, binge eating disorder, compulsive over-eating, impairedappetite regulation, metabolic syndrome, type 2 diabetes, dyslipidemia,atherosclerosis, or drug-induced obesity.
 8. The method of claim 1,wherein the human suffers from an over-eating disorder, bulimia nervosa,binge eating disorder, or compulsive over-eating.
 9. The method of claim1, wherein the drug-induced cardiovascular side effects are increasedheart rate, increased diastolic blood pressure, or increased systolicblood pressure, or a combination thereof.
 10. The method of claim 1,wherein the Tesofensine or the pharmaceutically acceptable salt thereofand the Metoprolol or the pharmaceutically acceptable salt thereof areadministered separately.
 11. The method of claim 1, wherein theTesofensine or the pharmaceutically acceptable salt thereof and theMetoprolol or the pharmaceutically acceptable salt thereof areadministered in a combined form.
 12. The method of claim 1, wherein theTesofensine or the pharmaceutically acceptable salt thereof isadministered once daily.
 13. The method of claim 1, wherein theMetoprolol or the pharmaceutically acceptable salt thereof isadministered once daily.
 14. The method of claim 1, wherein theTesofensin or the pharmaceutically acceptable salt thereof and theMetoprolol or the pharmaceutically acceptable salt thereof areadministered orally.
 15. The method of claim 14, wherein the Tesofensinor the pharmaceutically acceptable salt thereof and the Metoprolol orthe pharmaceutically acceptable salt thereof are each administered as atablet or capsule.
 16. The method of claim 1, wherein the human isadministered a pharmaceutically acceptable salt of Tesofensine and thepharmaceutically acceptable salt of Tesofensine is Tesofensine citrate.17. The method of claim 1, wherein the human is administered apharmaceutically acceptable salt of Metoprolol and the pharmaceuticallyacceptable salt of Metoprolol is metoprolol succinate.